We report population findings from newborn screening for biotinidase deficiency in California, representing over 2,000,000 newborns. The incidence of profound deficiency was 1/73,629, higher than in other reported populations. Out of 28 patients with profound biotinidase deficiency, 19 were of Hispanic descent, suggesting an increased frequency among this group. Of the 28 patients, 23 underwent mutation analysis of the BTD gene, with one common mutation, 528G>T, found in 43.3% of Hispanic alleles tested.

The WRKY gene family plays an important role in the development and stress responses in plants. As information is not available on the WRKY gene family in Musa species, genome-wide analysis has been carried out in this study using available genomic information from two species, Musa acuminata and Musa balbisiana. Analysis identified 147 and 132 members of the WRKY gene family in M. acuminata and M. balbisiana, respectively. Evolutionary analysis suggests that the WRKY gene family expanded much before the speciation in both the species. Most of the orthologs retained in two species were from the γ duplication event which occurred prior to α and β genome-wide duplication (GWD) events. Analysis also suggests that subtle changes in nucleotide sequences during the course of evolution have led to the development of new motifs which might be involved in neo-functionalization of different WRKY members in two species. Expression and cis-regulatory motif analysis suggest possible involvement of Group II and Group III WRKY members during various stresses and growth/development including fruit ripening process respectively.

The Drosophila glucoside xylosyltransferase Shams xylosylates Notch and inhibits Notch signaling in specific contexts including wing vein development. However, the molecular mechanisms underlying context-specificity of the shams phenotype is not known. Considering the role of Delta-Notch signaling in wing vein formation, we hypothesized that Shams might affect Delta-mediated Notch signaling in Drosophila. Using genetic interaction studies, we find that altering the gene dosage of Delta affects the wing vein and head bristle phenotypes caused by loss of Shams or by mutations in the Notch xylosylation sites. Clonal analysis suggests that loss of shams promotes Delta-mediated Notch activation. Further, Notch trans-activation by ectopically overexpressed Delta shows a dramatic increase upon loss of shams. In agreement with the above in vivo observations, cell aggregation and ligand-receptor binding assays show that shams knock-down in Notch-expressing cells enhances the binding between Notch and trans-Delta without affecting the binding between Notch and trans-Serrate and cell surface levels of Notch. Loss of Shams does not impair the cis-inhibition of Notch by ectopic overexpression of ligands in vivo or the interaction of Notch and cis-ligands in S2 cells. Nevertheless, removing one copy of endogenous ligands mimics the effects of loss shams on Notch trans-activation by ectopic Delta. This favors the notion that trans-activation of Notch by Delta overcomes the cis-inhibition of Notch by endogenous ligands upon loss of shams. Taken together, our data suggest that xylosylation selectively impedes the binding of Notch with trans-Delta without affecting its binding with cis-ligands and thereby assists in determining the balance of Notch receptor's response to cis-ligands vs. trans-Delta during Drosophila development.

In multicellular organisms, glycosylation regulates various developmental signaling pathways including the Notch pathway. One of the O-linked glycans added to epidermal growth factor-like (EGF) repeats in animal proteins including the Notch receptors is the xylose-xylose-glucose-O oligosaccharide. Drosophila glucoside xylosyltransferase (Gxylt) Shams negatively regulates Notch signaling in specific contexts. Since Shams adds the first xylose residue to O-glucose, its loss-of-function phenotype could be due to the loss of the first xylose, the second xylose or both. To examine the contribution of the second xylose residues to Drosophila Notch signaling, we have performed biochemical and genetic analysis on CG11388, which is the Drosophila homolog of human xyloside xylosyltransferase 1 (XXYLT1). Experiments in S2 cells indicated that similar to human XXYLT1, CG11388 can add the second xylose to xylose-glucose-O glycans. Flies lacking both copies of CG11388 (Xxylt) are viable and fertile and do not show gross phenotypes indicative of altered Notch signaling. However, genetic interaction experiments show that in sensitized genetic backgrounds with decreased or increased Notch pathway components, loss of Xxylt promotes Delta-mediated activation of Notch. Unexpectedly, we find that in such sensitized backgrounds, even loss of one copy of the fly Gxylt shams enhances Delta-mediated Notch activation. Taken together, these data indicate that while the first xylose plays a key role in tuning the Delta-mediated Notch signaling in Drosophila, the second xylose has a fine-tuning role only revealed in sensitized genetic backgrounds.

MADS box gene family is transcription factor gene family that is involved in growth and development of eukaryotes. In plants the MADS box gene family is mainly associated with floral meristem identity and flower development, apart from being involved in nearly all the phases of plant growth. The MADS box gene family has also been shown to be involved during fruit development and ripening. In this study the MADS box gene family from Musa balbisiana was identified and the divergence of this gene family between Musa balbisiana and Musa acuminata studied. A total of 97 MADS box genes were identified from the genome of Musa balbisiana. Phylogenetic analysis showed that the MbMADS box genes were categorised into type I (α and γ; the β group was not distinguishable) and type II groups (MIKCc and MIKC* and MIKCc was further divided into 13 subfamilies). The typeII group has the largest number of genes and also showed the most expansion which could be correlated with the whole genome duplications. There were significant differences in the MADS box genes from Musa acuminata and Musa balbisiana during evolution that can be correlated with different floral phenotype and fruit ripening pattern. The divergence of the MADS RIN genes in Musa balbisiana as compared to Musa acuminata might play an important role in the slow ripening of Musa balbisiana fruits.

Intestinal barrier dysfunction leads to inflammation and associated metabolic changes. However, the relative impact of gut bacteria versus non-bacterial insults on animal health in the context of barrier dysfunction is not well understood. Here, we establish that loss of Drosophila N-glycanase 1 (Pngl) in a specific intestinal cell type leads to gut barrier defects, causing starvation and JNK overactivation. These abnormalities, along with loss of Pngl in enterocytes and fat body, result in Foxo overactivation, leading to hyperactive innate immune response and lipid catabolism and thereby contributing to lethality. Germ-free rearing of Pngl mutants rescued their developmental delay but not lethality. However, raising Pngl mutants on isocaloric, fat-rich diets partially rescued lethality. Our data indicate that Pngl functions in Drosophila larvae to establish the gut barrier, and that the lethality caused by loss of Pngl is primarily mediated through non-bacterial induction of immune and metabolic abnormalities.

The homeodomain zipper family (HD-ZIP) of transcription factors is present only in plants and plays important role in the regulation of plant-specific processes. The subfamily IV of HDZ transcription factors (HD-ZIP IV) has primarily been implicated in the regulation of epidermal structure development. Though this gene family is present in all lineages of land plants, members of this gene family have not been identified in banana, which is one of the major staple fruit crops. In the present work, we identified 21 HDZIV encoding genes in banana by the computational analysis of banana genome resource. Our analysis suggested that these genes putatively encode proteins having all the characteristic domains of HDZIV transcription factors. The phylogenetic analysis of the banana HDZIV family genes further confirmed that after separation from a common ancestor, the banana, and poales lineages might have followed distinct evolutionary paths. Further, we conclude that segmental duplication played a major role in the evolution of banana HDZIV encoding genes. All the identified banana HDZIV genes expresses in different banana tissue, however at varying levels. The transcript levels of some of the banana HDZIV genes were also detected in banana fruit pulp, suggesting their putative role in fruit attributes. A large number of genes of this family showed modulated expression under drought and salinity stress. Taken together, the present work lays a foundation for elucidation of functional aspects of the banana HDZIV encoding genes and for their possible use in the banana improvement programs.

Nuclear genes encode most mitochondrial proteins, and their mutations cause diverse and debilitating clinical disorders. To date, 1,200 of these mitochondrial genes have been recorded, while no standardized catalog exists of the associated clinical phenotypes. Such a catalog would be useful to develop methods to analyze human phenotypic data, to determine genotype-phenotype relations among many genes and diseases, and to support the clinical diagnosis of mitochondrial disorders. Here we establish a clinical phenotype catalog of 174 mitochondrial disease genes and study associations of diseases and genes. Phenotypic features such as clinical signs and symptoms were manually annotated from full-text medical articles and classified based on the hierarchical MeSH ontology. This classification of phenotypic features of each gene allowed for the comparison of diseases between different genes. In turn, we were then able to measure the phenotypic associations of disease genes for which we calculated a quantitative value that is based on their shared phenotypic features. The results showed that genes sharing more similar phenotypes have a stronger tendency for functional interactions, proving the usefulness of phenotype similarity values in disease gene network analysis. We then constructed a functional network of mitochondrial genes and discovered a higher connectivity for non-disease than for disease genes, and a tendency of disease genes to interact with each other. Utilizing these differences, we propose 168 candidate genes that resemble the characteristic interaction patterns of mitochondrial disease genes. Through their network associations, the candidates are further prioritized for the study of specific disorders such as optic neuropathies and Parkinson disease. Most mitochondrial disease phenotypes involve several clinical categories including neurologic, metabolic, and gastrointestinal disorders, which might indicate the effects of gene defects within the mitochondrial system. The accompanying knowledgebase (http://www.mitophenome.org/) supports the study of clinical diseases and associated genes.

The BXD genetic reference population is a recombinant inbred panel descended from crosses between the C57BL/6 (B6) and DBA/2 (D2) strains of mice, which segregate for about 5 million sequence variants. Recently, some of these variants have been established with effects on general metabolic phenotypes such as glucose response and bone strength. Here we phenotype 43 BXD strains and observe they have large variation (-5-fold) in their spontaneous activity during waking hours. QTL analyses indicate that -40% of this variance is attributable to a narrow locus containing the aryl hydrocarbon receptor (Ahr), a basic helix-loop-helix transcription factor with well-established roles in development and xenobiotic metabolism. Strains with the D2 allele of Ahr have reduced gene expression compared to those with the B6 allele, and have significantly higher spontaneous activity. This effect was also observed in B6 mice with a congenic D2 Ahr interval, and in B6 mice with a humanized AHR allele which, like the D2 allele, is expressed much less and has less enzymatic activity than the B6 allele. Ahr is highly conserved in invertebrates, and strikingly inhibition of its orthologs in D. melanogaster and C. elegans (spineless and ahr-1) leads to marked increases in basal activity. In mammals, Ahr has numerous ligands, but most are either non-selective (e.g. resveratrol) or highly toxic (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)). Thus, we chose to examine a major environmental influence--long term feeding with high fat diet (HFD)--to see if the effects of Ahr are dependent on major metabolic differences. Interestingly, while HFD robustly halved movement across all strains, the QTL position and effects of Ahr remained unchanged, indicating that the effects are independent. The highly consistent effects of Ahr on movement indicate that changes in its constitutive activity have a role on spontaneous movement and may influence human behavior.

Phytoene synthase (PSY) regulates the first committed step of the carotenoid biosynthetic pathway in plants. The present work reports identification and characterization of the three PSY genes (TaPSY1, TaPSY2 and TaPSY3) in wheat (Triticum aestivum L.). The TaPSY1, TaPSY2, and TaPSY3 genes consisted of three homoeologs on the long arm of group 7 chromosome (7L), short arm of group 5 chromosome (5S), and long arm of group 5 chromosome (5L), respectively in each subgenomes (A, B, and D) with a similarity range from 89% to 97%. The protein sequence analysis demonstrated that TaPSY1 and TaPSY3 retain most of conserved motifs for enzyme activity. Phylogenetic analysis of all TaPSY revealed an evolutionary relationship among PSY proteins of various monocot species. TaPSY derived from A and D subgenomes shared proximity to the PSY of Triticum urartu and Aegilops tauschii, respectively. The differential expression of TaPSY1, TaPSY2, and TaPSY3 in the various tissues, seed development stages, and stress treatments suggested their role in plant development, and stress condition. TaPSY3 showed higher expression in all tissues, followed by TaPSY1. The presence of multiple stress responsive cis-regulatory elements in promoter region of TaPSY3 correlated with the higher expression during drought and heat stresses has suggested their role in these conditions. The expression pattern of TaPSY3 was correlated with the accumulation of β-carotene in the seed developmental stages. Bacterial complementation assay has validated the functional activity of each TaPSY protein. Hence, TaPSY can be explored in developing genetically improved wheat crop.

Azadirachta indica A. Juss, commonly known as Neem, is the reservoir of triterpenoids of economic importance. Metabolite analysis of different developmental stages of leaf and fruit suggests tissue-specific accumulation of the major triterpenoids in this important tree. Though biosynthesis of these complex molecules requires substrate flux from the isoprenoid pathway, enzymes involved in late biosynthetic steps remain uncharacterized. We established and analyzed transcriptome datasets from leaf and fruit and identified members of gene families involved in intermediate steps of terpenoid backbone biosynthesis and those related to secondary transformation leading to the tissue-specific triterpenoid biosynthesis. Expression analysis suggests differential expression of number of genes between leaf and fruit and probable participation in the biosynthesis of fruit-specific triterpenoids. Genome-wide analysis also identified members of gene families putatively involved in secondary modifications in late biosynthetic steps leading to the synthesis of highly oxygenated triterpenoids. Expression and molecular docking analyses suggest involvement of specific members of CYP450 family in secondary modifications for the biosynthesis of bioactive triterpenoids. This study generated rich genomic resource and identified genes involved in biosynthesis of important molecules, which will aid in the advancement of tools for functional genomics and elucidation of the biosynthesis of triterpenoid from this important tree.

Flavonoid biosynthesis is largely regulated at the transcriptional level due to the modulated expression of genes related to the phenylpropanoid pathway in plants. Although accumulation of different flavonoids has been reported in banana, a staple fruit crop, no detailed information is available on regulation of the biosynthesis in this important plant. We carried out genome-wide analysis of banana (Musa acuminata, AAA genome) and identified 28 genes belonging to 9 gene families associated with flavonoid biosynthesis. Expression analysis suggested spatial and temporal regulation of the identified genes in different tissues of banana. Analysis revealed enhanced expression of genes related to flavonol and proanthocyanidin (PA) biosynthesis in peel and pulp at the early developmental stages of fruit. Genes involved in anthocyanin biosynthesis were highly expressed during banana fruit ripening. In general, higher accumulation of metabolites was observed in the peel as compared to pulp tissue. A correlation between expression of genes and metabolite content was observed at the early stage of fruit development. Furthermore, this study also suggests regulation of flavonoid biosynthesis, at transcriptional level, under light and dark exposures as well as methyl jasmonate (MJ) treatment in banana.

Mitochondrial diseases are a clinically heterogenous group of disorders caused by respiratory chain dysfunction and associated with progressive, multi-systemic phenotype. There is no effective treatment or cure, and no FDA-approved drug for treating mitochondrial disease. To identify and characterize potential therapeutic compounds, we developed an in vitro screening assay and identified a group of direct AMP-activated protein kinase (AMPK) activators originally developed for the treatment of diabetes and metabolic syndrome. Unlike previously investigated AMPK agonists such as AICAR, these compounds allosterically activate AMPK in an AMP-independent manner, thereby increasing specificity and decreasing pleiotropic effects. The direct AMPK activator PT1 significantly improved mitochondrial function in assays of cellular respiration, energy status, and cellular redox. PT1 also protected against retinal degeneration in a mouse model of photoreceptor degeneration associated with mitochondrial dysfunction and oxidative stress, further supporting the therapeutic potential of AMP-independent AMPK agonists in the treatment of mitochondrial disease.

DNA polymorphic markers and self-defined ethnicity groupings are used to group individuals with shared ancient geographic ancestry. Here we studied whether ancestral relationships between individuals could be identified from metabolic screening data reported by the California newborn screening (NBS) program. NBS data includes 41 blood metabolites measured by tandem mass spectrometry from singleton babies in 17 parent-reported ethnicity groupings. Ethnicity-associated differences identified for 71% of NBS metabolites (29 of 41, Cohen's d > 0.5) showed larger differences in blood levels of acylcarnitines than of amino acids (P < 1e-4). A metabolic distance measure, developed to compare ethnic groupings based on metabolic differences, showed low positive correlation with genetic and ancient geographic distances between the groups' ancestral world populations. Several outlier group pairs were identified with larger genetic and smaller metabolic distances (Black versus White) or with smaller genetic and larger metabolic distances (Chinese versus Japanese) indicating the influence of genetic and of environmental factors on metabolism. Using machine learning, comparison of metabolic profiles between all pairs of ethnic groupings distinguished individuals with larger genetic distance (Black versus Chinese, AUC = 0.96), while genetically more similar individuals could not be separated metabolically (Hispanic versus Native American, AUC = 0.51). Additionally, we identified metabolites informative for inferring metabolic ancestry in individuals from genetically similar populations, which included biomarkers for inborn metabolic disorders (C10:1, C12:1, C3, C5OH, Leucine-Isoleucine). This work sheds new light on metabolic differences in healthy newborns in diverse populations, which could have implications for improving genetic disease screening.

AP2/ERF domain containing transcription factor super family is one of the important regulators in the plant kingdom. The involvement of AP2/ERF family members has been elucidated in various processes associated with plant growth, development as well as in response to hormones, biotic and abiotic stresses. In this study, we carried out genome-wide analysis to identify members of AP2/ERF family in Musa acuminata (A genome) and Musa balbisiana (B genome) and changes leading to neofunctionalisation of genes. Analysis identified 265 and 318 AP2/ERF encoding genes in M. acuminata and M. balbisiana respectively which were further classified into ERF, DREB, AP2, RAV and Soloist groups. Comparative analysis indicated that AP2/ERF family has undergone duplication, loss and divergence during evolution and speciation of the Musa A and B genomes. We identified nine genes which are up-regulated during fruit ripening and might be components of the regulatory machinery operating during ethylene-dependent ripening in banana. Tissue-specific expression analysis of the genes suggests that different regulatory mechanisms might be involved in peel and pulp ripening process through recruiting specific ERFs in these tissues. Analysis also suggests that MaRAV-6 and MaERF026 have structurally diverged from their M. balbisiana counterparts and have attained new functions during ripening.

Improved second-tier tools are needed to reduce false-positive outcomes in newborn screening (NBS) for inborn metabolic disorders on the Recommended Universal Screening Panel (RUSP).

Mitogen Activated Protein Kinase (MAPK) cascade is a fundamental pathway in organisms for signal transduction. Though it is well characterized in various plants, there is no systematic study of this cascade in tea.

PTEN-induced putative kinase 1 (PINK1) is a mitochondria-targeted kinase whose mutations are a cause of Parkinson's disease. We set out to better understand PINK1's effects on mitochondrial proteins in vivo. Using an unbiased phosphoproteomic screen in Drosophila, we found that PINK1 mediates the phosphorylation of MCAD, a mitochondrial matrix protein critical to fatty acid metabolism. By mimicking phosphorylation of this protein in a PINK1 null background, we restored PINK1 null's climbing, flight, thorax, and wing deficiencies. Owing to MCAD's role in fatty acid metabolism, we examined the metabolic profile of PINK1 null flies, where we uncovered significant disruptions in both acylcarnitines and amino acids. Some of these disruptions were rescued by phosphorylation of MCAD, consistent with MCAD's rescue of PINK1 null's organismal phenotypes. Our work validates and extends the current knowledge of PINK1, identifies a novel function of MCAD, and illuminates the need for and effectiveness of metabolic profiling in models of neurodegenerative disease.

Ethylene signal transduction in plants is conducted by the two-component system (TCS) which consists of histidine kinase (HK), histidine phosphotransferase (HPT) and response regulators (RRs). This system plays an important role in signal transduction during various cellular processes, including fruit ripening and response to multiple environmental cues. Though members of TCS have been identified in a few plants, no detailed analysis has been carried out in banana.

Fringe glycosyltransferases differentially modulate the binding of Notch receptors to Delta/DLL versus Serrate/Jagged ligands by adding GlcNAc to O-linked fucose on Notch epidermal growth factor-like (EGF) repeats. Although Notch has 22 O-fucosylation sites, the biologically relevant sites affecting Notch activity during animal development in vivo in the presence or absence of Fringe are not known. Using a variety of assays, we find important roles in Drosophila Notch signaling for GlcNAc-fucose-O glycans on three sites: EGF8, EGF9, and EGF12. O-Fucose monosaccharide on EGF12 (in the absence of Fringe) is essential for Delta-mediated lateral inhibition in embryos. However, wing vein development depends on the addition of GlcNAc to EGF8 and EGF12 by Fringe, with a minor contribution from EGF9. Fringe modifications of EGF8 and EGF12 together prevent Notch from cis-inhibiting Serrate, thereby promoting normal wing margin formation. Our work shows the combinatorial and context-dependent roles of GlcNAc-fucose-O glycans on these sites in Drosophila Notch-ligand interactions.